Cell size and mutual cell adhesion

Summary HeLa cells harvested from density-inhibited or fast growing suspension cultures, were incubated in NaCl solutions of different tonicity. Cell size enlargement produced by hypotonicity is accompanied by an increased sedimentation rate of the density-inhibited cells, whereas no appreciable change is observed in the sedimentation rate of fast growing cells. Hypotonicity also has no effect on the sedimentation rate of density-inhibited cells which previously had been treated with neuraminidase or trypsin. It is shown that the effect of hypotonicity on density-inhibited cells cannot be ascribed to release of cell surface sialic acids during hypotonic incubation. Several arguments are presented which indicate that the changes in sedimentation rate, as measured in the rotating suspension system, are not the direct consequence of the alterations in cell size, but rather must be attributed to differences in intercellular adhesiveness resulting from the size alterations. Analogous changes in intercellular adhesiveness and cell size are shown to occur during growth in isotonic suspension culture. The results can be explained by assuming that changes in cell size affect the intercellular adhesiveness by modifying the extent to which cell surface sialic acids counteract adhesion.     

Structure, function, and fate of the BlaR signal transducer involved in induction of beta-lactamase in Bacillus licheniformis.

The membrane-spanning protein BlaR is essential for the induction of beta-lactamase in Bacillus licheniformis. Its nature and location were confirmed by the use of an antiserum specific for its carboxy-terminal penicillin sensor, its function was studied by genetic dissection, and the structure of the penicillin sensor was derived from hydrophobic cluster analysis of the amino acid sequence by using, as a reference, the class A beta-lactamases with known three-dimensional structures. During the first 2 h after the addition of the beta-lactam inducer, full-size BlaR, bound to the plasma membrane, is produced, and then beta-lactamase is produced. By 2 h after induction, BlaR is present in various (membrane-bound and cytosolic) forms, and there is a gradual decrease in beta-lactamase production. The penicillin sensors of BlaR and the class D beta-lactamases show strong similarities in primary structures. They appear to have the same basic spatial disposition of secondary structures as that of the class A beta-lactamases, except that they lack several alpha helices and, therefore, have a partially uncovered five-stranded beta sheet and a more readily accessible active site. Alterations of BlaR affecting conserved secondary structures of the penicillin sensor and specific sites of the transducer annihilate beta-lactamase inducibility.     

MicroFilament Analyzer,an image analysis tool for quantifying fibrillar orientation,reveals changes in microtubule organization during gravitropism

Image acquisition is an important step in the study of cytoskeleton organization. As visual interpretations and manual measurements of digital images are prone to errors and require a great amount of time, a freely available software package named MicroFilament Analyzer (MFA) was developed. The goal was to provide a tool that facilitates high‐throughput analysis to determine the orientation of filamentous structures on digital images in a more standardized, objective and repeatable way. Here, the rationale and applicability of the program is demonstrated by analyzing the microtubule patterns in epidermal cells of control and gravi‐stimulated Arabidopsis thaliana roots. Differential expansion of cells on either side of the root results in downward bending of the root tip. As cell expansion depends on the properties of the cell wall, this may imply a differential orientation of cellulose microfibrils. As cellulose deposition is orchestrated by cortical microtubules, the microtubule patterns were analyzed. The MFA program detects the filamentous structures on the image and identifies the main orientation(s) within individual cells. This revealed four distinguishable microtubule patterns in root epidermal cells. The analysis indicated that gravitropic stimulation and developmental age are both significant factors that determine microtubule orientation. Moreover, the data show that an altered microtubule pattern does not precede differential expansion. Other possible applications are also illustrated, including field emission scanning electron micrographs of cellulose microfibrils in plant cell walls and images of fluorescent actin.     

Regulation of the transient receptor potential channel TRPM3 by phosphoinositides

The transient receptor potential (TRP) channel TRPM3 is a calcium-permeable cation channel activated by heat and by the neurosteroid pregnenolone sulfate (PregS). TRPM3 is highly expressed in sensory neurons, where it plays a key role in heat sensing and inflammatory hyperalgesia, and in pancreatic β cells, where its activation enhances glucose-induced insulin release. However, despite its functional importance, little is known about the cellular mechanisms that regulate TRPM3 activity. Here, we provide evidence for a dynamic regulation of TRPM3 by membrane phosphatidylinositol phosphates (PIPs). Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P₂) and ATP applied to the intracellular side of excised membrane patches promote recovery of TRPM3 from desensitization. The stimulatory effect of cytosolic ATP on TRPM3 reflects activation of phosphatidylinositol kinases (PI-Ks), leading to resynthesis of PIPs in the plasma membrane. Various PIPs directly enhance TRPM3 activity in cell-free inside-out patches, with a potency order PI(3,4,5)P₃ > PI(3,5)P₂ > PI(4,5)P₂ ≈ PI(3,4)P₂ >> PI(4)P_. Conversely, TRPM3 activity is rapidly and reversibly inhibited by activation of phosphatases that remove the 5-phosphate from PIPs. Finally, we show that recombinant TRPM3, as well as the endogenous TRPM3 in insuloma cells, is rapidly and reversibly inhibited by activation of phospholipase C–coupled muscarinic acetylcholine receptors. Our results reveal basic cellular mechanisms whereby membrane receptors can regulate TRPM3 activity.     

Sequence of a gene encoding a (poly ManA) alginate lyase active on Pseudomonas aeruginosa alginate

Abstract To overcome problems associated with Western blotting of denatured proteins, we have used quantitative immunoelectrophoretic techniques to perform functional analysis of the Neisseria gonorrhoeae common antigen. Using these techniques, we show (a) that Neisseria gonorrhoeae expresses an antigen that is cross-reactive with the common antigen of Pseudomonas aeruginosa and Legionella micdadei and with the GroEl-like protein of Chlamydia , and (b) that this N. gonorrhoeae common antigen has lectin-like activity and can be precipitated with three different sugars immobilized on agarose beads: α- d -glucosamine, maltose and fucose.     

A short course of infusion of a hydrogen sulfide-donor attenuates endotoxemia induced organ injury via stimulation of anti-inflammatory pathways,with no additional protection from prolonged infusion

Organ failure is associated with increased mortality and morbidity in patients with systemic inflammatory response syndrome. Previously, we showed that a short course of infusion of a hydrogen sulfide (H₂S) donor reduced metabolism with concurrent reduction of lung injury. Here, we hypothesize that prolonged H₂S infusion is more protective than a short course in endotoxemia with organ failure. Also, as H₂S has both pro- and anti-inflammatory effects, we explored the effect of H₂S on interleukin production.Endotoxemia was induced by an intravenous bolus injection of LPS (7.5 mg/kg) in mechanically ventilated rats. H₂S donor NaHS (2 mg/kg) or vehicle (saline) was infused and organ injury was determined after either 4 or 8 h. A short course of H₂S infusion was associated with reduction of lung and kidney injury. Prolonged infusion did not enhance protection. Systemically, infusion of H₂S increased both the pro-inflammatory response during endotoxemia, as demonstrated by increased TNF-α levels, as well as the anti-inflammatory response, as demonstrated by increased IL-10 levels. In LPS-stimulated whole blood of healthy volunteers, co-incubation with H₂S had solely anti-inflammatory effects, resulting in decreased TNF-α levels and increased IL-10 levels. Co-incubation with a neutralizing IL-10 antibody partly abrogated the decrease in TNF-α levels. In conclusion, a short course of H₂S infusion reduced organ injury during endotoxemia, at least in part via upregulation of IL-10.     

Parvalbumins from the lungfish (Protopterus dolloi).

Five parvalbumins have been isolated from the white muscles of the lungfish. They can be divided into two sub families showing typical amino acid compositions, C-terminal amino acid residues, peptide maps and immuno-reactivity. The red muscles including the cardiac muscle also contain parvalbumins in amounts roughly inversely related to the concentration of myoglobin in the muscle. Parvalbumins have also been detected in the brain and kidney.